mol15 - Lecture 15 - Transcriptional regulation! Continuing...

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Lecture 15 - Transcriptional regulation Parts & Processes Continuing Chapter 7 α -amanatin insensitive clarification from L14 PolII is sensitive to α -amanatin S1 analysis to measure mRNA genomic globin clone In HeLa cells (non erythro) 4.7 kB Enhancer function A new way to measure DNaseI footprinting Gel shift (EMSA) assays To detect protein::DNA binding To detect functional domains Deletion series and in vivo assay
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MCB 7-8 three distinct eukaryotic RNA polymerases Pol I - nucleolar, pre-rRNA (28S, 5.8S and 18S) Pol III - non nucleolar tRNAs, 5S rRNA, and other small RNAs Pol II -- transcribes protein-coding genes (mRNAs) α -amanatin insensitive Results: “death cap” mushroom – A. phalloides toxin is an 8 amino acid cyclic peptide clarification from L14 clarification from L14
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MCB 7-16 general anatomy of eukaryotic transcriptional control sequences How do we fnd what binds the sequences to regulate RNA polymerase and transcription?
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identifying transcriptional regulators (txn ʻ factors ʼ ) regulators are modular: distinct functional domains ( a gallery of motifs) combinatorial control multiprotein complexes enhancers Pol II initiation complex itself Transcriptional Regulation mechanisms of repression and activation revisiting chromatin Factor: one or more proteins having a specifed activity
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regulating initiation by protein-DNA binding The general theme mRNA gene “+1” start site negative numbers “upstream” positive numbers “downstream” two techniques for detecting protein-DNA binding DNaseI ʻ footprinting ʼ Gel-shift assays (retardation, EMSA) the conventions Biochemical and Genetic approaches have revealed regulators that bind speciFc sequences end Tuesday
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DNase I footprinting reveals speciFc binding proteins * labeled DNA +/- proteins digest with DNaseI remove protein, denature DNA electrophorese, autoradiograph * MCB 7-18
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DNase I footprinting reveals speciFc binding proteins [NaCl] (Assay to monitor puriFcation) MCB 7-17
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mol15 - Lecture 15 - Transcriptional regulation! Continuing...

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